Classical pharmacological studies have defined three classes of opiate receptors-the delta, kappa and mu. The delta receptors bind with high affinity to enkephalins. Prolonged exposure to opiates cause desensitization and down-regulation of the delta receptor. This has been proposed to play a significant role in the development of opiate tolerance and dependence. However, little is known regarding the molecular basis for desensitization and down-regulation of the receptor. In this grant application we propose to address this issue. The cloning of the delta opiate receptor cDNA from NG1O8-15 cells has confirmed that these receptors belong to the 'G-protein coupled receptor' family. Accordingly, the opiate receptors share many structural features with the G-protein coupled receptors including rhodopsin and beta adrenergic receptor. Many posttranslational modifications are found to regulate these other receptors as revealed by mutational analyses. Recently phosphorylation of the beta adrenergic receptor and rhodopsin has been implicated in desensitization of these receptors. The objective of the studies proposed in this grant application is to examine the involvement of phosphorylation in the regulation of opiate receptor. The delta opiate receptor will be subjected to in vitro and in situ phosphorylation and the phosphorylated receptor will be immunoaffinity purified. Phosphopeptides will be generated, isolated and characterized. The phosphopeptide maps obtained from the desensitized receptors will be compared to the normal receptor. The involvement of specific phosphorylation sites on the desensitization will be analyzed by site-specific mutagenesis. The effect of mutations on the desensitization and down regulation will be examined. The studies described in this grant application will provide an important insight towards the role of phosphorylation in the development of opiate tolerance and dependence.